Our focus is on cholesterol-independent and dependent mechanisms whereby statins afford neuroprotection to cells undergoing oxidative stress. Statins reduce cholesterol levels but also they have cholesterol- independent mechanisms of action. Neuroprotective effects of statins could involve multiple pathways.We have discovered that statins altered expression of genes involved in apoptosis, cell growth, signaling and trafficking in the murine cerebral cortex. A novel finding was that simvastatin increased gene expressionand protein levels of Bcl-2 in vivo and in vitro. Bcl-2 and anti-apoptotic members of the Bcl-2 family play apivotal role in neuronal cell survival. Simvastatin also significantly increased ET-1 expression levels whose product is the precursor for the ET-1 protein. ET-1 is involved in transcriptional activation of Bcl-2. Preliminary data revealed that when Bcl-2 protein levels were experimentally reduced the neuroprotective effects of simvastatin to an Ap challenge were eliminated. We hypothesizethat: Simvastatin has both cholesterol- independent and-dependent mechanisms of action that are neuroprotective. Neuroprotective mechanisms are due to regulation of apoptosis by Bcl-2 family members, including the transcriptional regulation of Bcl-2 by ET-1/calcineurin/NFAT-dependent pathways and inhibition of Rac1 geranylgeranylation. These hypotheses will be testing primarily in mouse primary cortical and hippocampal neurons. Aim 1. Determine Bcl-2 and Bax gene expression and protein levels in neurons pretreated with simvastatin and challenged by A(Jor NMDA. Determine if simvastatin-induced increasein Bcl-2 inhibits translocation of apoptosis -inducing factor induced by Ap or NMDA from the mitochondria to the nucleus in neurons. Examine whether suppression of Bcl-2 gene expression or inactivation of the protein diminishes neuroprotective effects of simvastatin in neuroblastoma cells treated with Ap or NMDA. Aim 2. Determine if ET-1 increases Bcl-2 gene expression and protein levels in neurons challenged with Ap or NMDA. Evaluate if ET-1/calcineurin induces NFAT to the nucleus, binding to the NFAT sites on the Bcl-2 promoter region and increases Bcl-2 gene expression in neurons. Determine if suppression of ET-1 expression reduces simvastatin-induced stimulation of Bcl-2 gene expression and protein levels and neuroprotection when challenged by Ap or NMDA in human neuroblastoma cells. Aim 3. Determine if lowering cholesterol levels directly by cyclodextrin increases Bcl-2 and ET-1 gene expression andprotein levels in neurons and if neuroprotective. Evaluate if Bcl-2 and ET-1 gene expression and protein levels and neuroprotection are increased when farnesylation and geranylgeranylation are inhibited by non-statin inhibitors in neurons. Determine if simvastatin-induced stimulation of Bcl-2 and ET-1 gene expression and protein levels and neuroprotection can be inhibited by isoprenoids in neurons.